Carbo-Br to inhibit [125I]CYP binding was shown in Figure 2-
1B. In the absence of Gpp(NH)p, the IC50 value was 3.3 0.3

nM and the addition of Gpp(NH)p only shifted the IC50 value

2.2-fold to 7.6 0.3 nM.

Figure 2-2 showed a representative Scatchard plot of

[125I]CYP binding after pretreatment of reticulocyte mem-

branes with Carbo-Br or Carbo-Am. As Iso is easily washed

from membranes, no control for Iso pretreatment was neces-

sary. Membranes incubated with 25 nM Carbo-Br for 30 min at

320C followed by four washes had an 82% reduction of specific
[125I]CYP binding sites with no change in the KD value of

[125I]CYP for the remaining receptors (control, 14 pM; Carbo-

Br-treated, 12 pM). When 100 gM Gpp(NH)p was added during

the pretreatment period, the loss of specific binding sites
was reduced to 65% with no alteration of the KD value (13 pM)

as compared to the control for the remaining receptors

(Figure 2-2A). Figure 2-2B showed a Scatchard plot after

Figure 2-1. Inhibition of specific [125I]CYP binding in rat
reticulocyte membranes by Iso, Carbo-Am (A) and Carbo-Br (B).
Membranes were incubated with buffer at pH 7.4, 30 pM
[125I]CYP, the indicated concentrations of (A) Iso (squares),
Carbo-Am (circles) or (B) Carbo-Br (circles) and without
(open symbols) or with (closed symbols) 100 JiM Gpp(NH)p for
45 min at 360C. In the Iso competition assays, 0.1%
ascorbate was also present. At the end of the incubation,
the specific binding was determined as described in the
"Methods" section. Each point on the graph is the mean of
three determinations assayed in triplicate. Control
[125I]CYP binding ranged from 524 to 583 fmol/mg protein.

0-%

n

0
-C

LO
cm

C
0

n
a-
U
"-
H
If
TJ

Ligand (-Log M)

Figure 2-2. Scatchard plot of specific [125I]CYP binding to
rat reticulocyte membranes after treatment with (A) Carbo-Br
or (B) Carbo-Am. In A, membranes were incubated in buffer
without (open circles) and with (closed circles) 25 nM Carbo-
Br or with 25 nM Carbo-Br plus 100 pM Gpp(NH)p (squares) for
30 min at 320C. In B, membranes were incubated without (open
circles) and with (closed circles) 0.1 pM Carbo-Am or with
0.1 JM Carbo-Am plus 100 IM Gpp(NH)p (squares) for 30 min at
32 oC. At the end of the incubation, the membranes were
washed four times with buffer and assayed with 3 to 100 pM
[125I]CYP as described in the "Methods" section. The data
were plotted as the ratio of the amount of specifically bound
ligand (pmol/mg protein) to free ligand (pmol/l) versus the
amount of specifically bound ligand/mg protein. Data points
were the mean of triplicate determinations and are
representative of three experiments.

0
T-
x
320-

O X
10
40
50-

40

100 300 500
125CYP Bound(f mg protein)
c20

i 10-

.0
100 300 500
EC125JCYP Bound (fmdl/mg protein)

pretreatment of reticulocyte membranes with 0.1 g1M Carbo-Am

for 30 min at 320C followed by four wash cycles. The Carbo-

Am-treated membranes showed a 73% reduction in specific
[125I]CYP binding with no change in the KD value of [125I]CYP

for the remaining receptors (control, 8 pM; Carbo-Am-

treated,10.4 pM). Inclusion of 100 JM Gpp(NH)p in the

preincubation reduced the Carbo-Am-induced receptor loss to

51% with no change in the KD value for [125I]CYP (10.8 pM).

In one other series of experiments, the ability of various

doses of the two derivatives to induce a receptor loss was

determined. Incubation of reticulocyte membranes at 320C for

30 min with 10, 1, 0.1 and 0.01 JM of Carbo-Br followed by

four membrane wash cycles reduced specific [125I]CYP binding

by 97, 93, 86 and 80%, respectively. In contrast, using the

same concentrations of Carbo-Am, the receptor content was

reduced by 73, 73, 72 and 62%, respectively.

Table 2-1 showed the effects of several treatments on

the ability of the carbostyril derivatives to bind to the

beta-adrenoreceptor in reticulocyte membranes. Preincubation

of membranes with nadolol (10 gM) followed by washing did not

reduce specific [125I]CYP binding indicating that this beta-

antagonist is easily washed off the receptor and out of the

membranes (unlike propranolol). Furthermore, membrane

heating at 450C for 30 min had no significant effect on the

receptor. Preincubation with Carbo-Am (1 JM) followed by

membrane washing, reduced the binding by 73%, which could be

Table 2-1. Effects of nadolol and heat treatment on the
Carbo-Am and Carbo-Br-induced receptor loss in reticulocyte
membranes.

[125I]CYP Bound
Pretreatment A Pretreatment B (%Control)

----- ---- 100

Nadolol (10 gM) ------- 110 6

----- 450C 97 4

Carbo-Am (1 LM) ----- 27 3

Carbo-Am (1 gM) plus
Nadolol (10 rM) ----- 87 5

Carbo-Am (1 iM) Nadolol (10 gM) 29 6

Carbo-Am (1 IgM) 450C plus Nadolol (10 pM) 82 4

Carbo-Br (1 RM) ----- 12 4

Carbo-Br (1 gM) plus
Nadolol (10 gM) ----- 96 2

Carbo-Br (1 gM) 450C plus Nadolol (10 IM) 16 2

Carbo-cysteine (1 iM) ------ 25 4

Carbo-cysteine (1 IM) 450C plus Nadolol (10 pM) 86 6

In pretreatment A, membrane protein was incubated with
buffer and the additions indicated for 30 min at 320C. At
the end of the incubation, the membranes were washed four
times and assayed with [125I]CYP (100 pM). Alternatively, at
the end of pretreatment A, the membranes were washed once,
resuspended in 50 mM sodium phosphate buffer plus the
additions under pretreatment B and incubated for 30 min at
45C or at 320C for 20 min in the presence of 10 iM nadolol.
At the end of the incubation, the membranes were washed four
times with ice-cold buffer and assayed with [125I]CYP at 100
pM. Carbo-cysteine is Carbo-Br (0.5 mM) reacted for 18 hr at
25C with cysteine (2 mM) and diluted to the final concentra-
tion indicated. Values are the mean of triplicate determin-
ations S.D, n=3-4.

largely prevented (87% of control) by concurrent incubation

with 10 gM nadolol. However, no receptor recovery occurred

if the membranes were incubated with 10 .LM nadolol after the

Carbo-Am pretreatment. The initial Carbo-Am-induced loss of

receptors could be substantially recovered (82% of control)

by subsequent membrane heating at 450C in the presence of

nadolol. Table 2-1 also showed that membranes pretreated

with Carbo-Br (1 pM) followed by washing had an 88% decrease

in specific [125I]CYP binding which was largely attenuated by

concurrent pretreatment with 10 pM nadolol. When membranes

were pretreated with Carbo-Br, washed and then incubated with

nadolol at 450C for 30 min followed by washing, there was

little reversal (<10%) of the lost specific binding sites.

In contrast, when a solution of Carbo-Br was incubated with

cysteine (250C, 18 hr), diluted to 1 gM and incubated with

membranes followed by washing, there was a 75% loss of

binding sites. These lost sites were largely recovered (86%

of control) if the pretreated membranes were further

incubated with nadolol at 450C for 30 min followed by

washing. A diluted sample of cysteine alone had no effect on

[125I]CYP binding.

Current evidence indicated that in the absence of a

guanine nucleotide, beta-agonists promoted the formation of a

ternary complex composed of the agonist, the beta-adreno-

receptor and Ns. Agonist binding in the complex was of high

affinity. In the presence of a guanine nucleotide, the

complex destablized and agonist affinity decreased (De Lean

et al., 1980; Kent et al., 1980). To explore if the small

Gpp(NH)p-induced potency shift for the Carbo-Am was due to a
tightly bound receptor-Ns complex resistant to guanine

Gpp(NH)p to 1182 20 nM in the presence of 100 gM Gpp(NH)p.
After treatment of membranes for 45 min at 500C, the IC50

value for Iso in the absence of Gpp(NH)p was 750 57 nM.

The IC50 value for Carbo-Am in control membranes and in the

absence of Gpp(NH)p was 7.3 0.3 nM which was increased

Figure 2-3. Inhibition of specific [125I]CYP binding in rat
erythrocyte (A) and reticulocyte (B) membranes by Iso and
Carbo-Am. In A, erythrocyte membranes were incubated with
buffer, 30 pM [125I]CYP and the indicated concentrations of
Iso (squares), Iso plus 100 JM Gpp(NH)p (triangles), Carbo-Am
(open circles) or Carbo-Am plus 100 p!M Gpp(NH)p (closed
circles) for 45 min at 360C. In B, control reticulocyte
membranes were incubated with buffer, 30 pM [125I]CYP and the
indicated concentration of Iso (open squares) and Iso plus
100 JM Gpp(NH)p (closed squares), Carbo-Am (open circles) or
Carbo-Am plus 100 IM Gpp(NH)p (closed circles), for 45 min at
360C. In addition, reticulocyte membranes in 50 mM sodium
phosphate buffer at pH 7.4 were incubated at 500C for 45 min,
washed 1 time in buffer and the competition assay carried out
with the indicated concentration of Iso (closed triangles)
and Carbo-Am (open triangles). In all assays utilizing Iso,
0.1% ascorbate was also present. At the end of the incuba-
tions, the specific binding was determined as described in
the "Methods" section. Each point on the graph was the mean
of three determinations assayed in triplicate. The control
[125I]CYP binding values were 39 7, 622 13 and 644 12
fmol/mg protein for the erythrocyte, control reticulocyte and
heat-treated reticulocyte membranes, respectively.

0
.Co
c-

10(
45
5 8'

6(

o
4(
0..
U
-l
H 2(
if)
C\1
Ti_

Ligand (-Log M)

to 26 1.1 nM in the presence of Gpp(NH)p. After heat

treatment, the IC50 value in the absence of Gpp(NH)p was 21

1.5 nM. The IC50 values of the Carbo-Am in the presence of

Gpp(NH)p in control reticulocytes (26 nM) was virtually

identical to the heat-treated membranes (21 nM) in the

absence of Gpp(NH)p and erythrocyte membranes both in the

presence or absence of Gpp(NH)p (27-29 nM).

Effects of the carbostyril derivatives on reticulocyte

adenylate cyclase activity. Figure 2-4 showed the ability of

Iso and the two carbostyril derivatives to stimulate cAMP

formation. The concentration that produced half-maximal

formation was 8.2 2.1, 17.8 3.1 and 241 17 nM for

Carbo-Br, Carbo-Am and Iso, respectively. In addition, the

maximal formation of cAMP was the same for all 3 compounds.

Unlike Iso, both of the carbostyril derivatives appear to be

quite stable agonists: even after several weeks in solution

they retained full potency and efficacy to stimulate

adenylate cyclase. Table 2-2 showed the partial specificity

of Carbo-Br in the adenylate cyclase system. In reticulocyte

membranes, Forskolin (1 gM) stimulated cAMP production by

17.9-fold. When Iso (10 gM) or Carbo-Br (10 IM) was added in

addition to Forskolin, the fold stimulation for both was 23.

Using GH3 cell membranes, forskolin (1 IM) stimulated cAMP

formation 15.9-fold. However, Carbo-Br (10 gM) did not

stimulate cAMP formation above the basal level. Membranes

from GH3cells did not appear to contain beta-adrenoreceptors

32

200
C

01160-
E
o,

E

1 80 -

4O
40-

10 9 8 7 6 5 4
Ligand (-LogM)

Figure 2-4. Stimulation of reticulocyte adenylate cyclase
activity by Iso, Carbo-Am and Carbo-Br. Cyclase activity was
determined by incubating membrane protein (35 gg) in buffer
pH 7.4 containing 500 lM GTP, the indicated concentration of
Iso (squares), Carbo-Am (closed circles) and Carbo-Br (open
circles) for 10 min at 320C. Other standard cyclase assay
components and the determination of the cAMP content were
performed as described in the "Methods" section. Each data
point is the mean S.D., n = 3. Basal activity in the
presence of GTP was 17 4 pmol/min/mg protein and was
subtracted from the stimulated values.

Membrane protein (reticulocytes, 35 gg; GH3, 50 gg) was
incubated with the standard cyclase assay components and the
additions indicated for 10 min at 320C. At the end of the
incubation, the cAMP formed was determined as described under
"Experimental Procedures". Values are the means of tripli-
cate determinations S.E., n=3-4.

aSignificantly different from the Forskolin group (p <0.01).

as no specific [125I]CYP (3-100 pM) binding was detectable

(data not shown).

The time course of cAMP formation in reticulocyte

membranes induced by Iso (10 1M), Carbo-Br (1 !LM) and Carbo-

Am (1 1M) was shown in Figures 2-5A, B and C, respectively.

Cyclic AMP formation in the presence of all three compounds

was linear for at least 14 min of incubation. When propran-

olol (20 1M) was added after 7 min of incubation, the Iso-

stimulated formation of cAMP was blocked (Figure 2-5A).

However, when propranolol (20 J!M) was added after 7 min of

incubation with Carbo-Br (Figure 2-5B) or Carbo-Am (Figure 2-

5C), no effect on the rate of cAMP formation was observed.

In contrast, if propranolol was added at time zero with the

three compounds, then cAMP formation by all three was largely

blocked. Figure 2-6 showed the time course of Carbo-Am and

Carbo-Br-induced receptor loss in reticulocyte membranes.

Incubations were carried out at 320C with 1 gM of both com-

pounds plus all of the additions used in the assay of adeny-

late cyclase activity. At various times during the

incubation, membrane samples were washed four times and

assayed for beta-adrenoreceptors. After 2 min of incubation,

specific [125I]CYP binding was decreased by 51 and 61% in

membranes incubated with Carbo-Am and Carbo-Br, respectively.

This loss continued slowly until, by the end of 12 min,

specific binding sites had decreased 61% in the Carbo-Am

treated membranes and 75% in those treated with

Figure 2-5. Time course of reticulocyte adenylate cyclase
activation by Iso (A), Carbo-Am (B) and Carbo-Br (C).
Cyclase activity was determined by incubating membrane
protein (35 gg) in buffer containing 500 pM GTP, 10 JIM Iso
(A), 1 gLM Carbo-Br (B) or 1 LIM Carbo-Am (C) at 320C.
Activity was determined with the agonists alone (open
circles), agonists plus 20 pIM propranolol added at time 0
(squares) or after 7 min of incubation with the agonists
alone, propranolol (20 AM) was added (closed circles). The
arrow indicated the time when propranolol was added. Other
standard cyclase components and the determination of the cAMP
content were as described in the "Methods" section. Each
data point was the mean of triplicate determinations and was
representative of four experiments. Basal cAMP formation in
the presence of GTP alone was subtracted from the stimulated
values.

Time (min)

2 80
C
0
U
60-

0

H
S20
(,1
c"u
LTJ

I I I I I I
0 2 4 6 8 10 12
Time (min)

Figure 2-6. Time course of specific [125I]CYP binding loss
by Carbo-Am and Carbo-Br in reticulocyte membranes. Membrane
protein (0.26 mg/ml) was incubated in buffer containing 1.6
mM ATP, 500 IM GTP, 1.0 mM EGTA, 10 mM theophylline, 0.1%
BSA, creatine phosphokinase (67 units/ml), 2.5 mM phospho-
creatine and without (squares) and with 1 pM Carbo-Am (closed
circles) or 1 gM Carbo-Br (open circles) at 320C. At the
times indicated, samples were removed, the membranes washed
four times with ice-cold buffer and assayed with 100 pM
[125I]CYP as described in the "Methods" section. Data points
are the mean of three determinations. The control [125I]CYP
binding values were 566 17 fmol/mg of protein in the Carbo-
Am experiments and 575 25 fmol/mg protein in the Carbo-Br
experiments.

Carbo-Br. Less than 5% of the binding sites were lost over

the 12 min incubation period in control membranes.

Discussion

The data from this study showed that both of the

carbostyril derivatives were highly potent beta-adrenergic

agonists. The Carbo-Am and Carbo-Br compounds were 14- and

29-fold, respectively, more potent than Iso in stimulating

adenylate cyclase activity. That enzyme activation by the

carbostyril derivatives was mediated through the beta-adreno-

receptor rather than a nonspecific effect was indicated by

two lines of evidence. First, concurrent addition of

propranolol, a beta-antagonist, blocked the enzyme activation

by both compounds. Second, the enzyme in GH3 cell membranes

was vigorously stimulated by forskolin but not by Carbo-Br.

These membranes did not contain any detectable beta-adreno-

receptors (Henneberry et al., 1986). The observation that

both of the carbostyril congeners produced the same maximal

stimulation of adenylate cyclase activity as Iso indicated

that both compounds were full beta-agonists.

Current evidence has suggested that a beta-agonist, the

beta-adrenoreceptor and Ns interact to form a ternary complex

(Citri and Schramm, 1980; De Lean et al., 1980; Kent et al.,

1980; Limbird et al., 1980b). When a guanine nucleotide has

bound to Ns, it destabilized the ternary complex, causing Ns

to dissociate from the receptor and reduced the receptor

affinity for the agonist which can then dissociated from the

receptor. Thus, in the absence of a guanine nucleotide, a

substantial fraction of the beta-adrenoreceptors showed high

affinity for the receptor ternaryy complex formation) whereas

in the presence of a guanine nucleotide all of the receptors

showed an agonist low affinity binding state (Lefkowitz et

al., 1976; Maguire et al., 1976; De Lean et al., 1980). The

initial ternary complex formation appeared to be a necessary

prerequisite for a beta-agonist to stimulate adenylate

cyclase activity (De Lean et al., 1980; Kent et al., 1980).

From competition studies in the absence of guanine nucle-

otide, the Carbo-Am and Carbo-Br compounds were 8- and 14-

fold more potent than Iso. In the presence of Gpp(NH)p,

however, the Carbo-Am and Carbo-Br were 39- and 106-fold

(respectively) more potent than Iso. This substantial

increase in the difference in potency between the carbostyril

derivatives and Iso in the presence of guanine nucleotides

was apparently due to the large Gpp(NH)p-induced decrease in

the affinity of Iso for the receptor (17-fold) whereas

Gpp(NH)p reduced the potency of the carbostyril compounds

only slightly (2- to 3.5-fold).

It has been reported that there is a direct relationship

between the ability of a beta-agonist to stimulate maximally

adenylate cyclase activity (intrinsic activity) and the

Gpp(NH)p-induced shift in agonist affinity (Lefkowitz et al.,

1976; Kent et al., 1980). Full agonists have large affinity

shifts whereas partial agonists have small affinity shifts.

Since both of the carbostyril congeners act as full agonists

with relatively small Gpp(NH)p-induced potency shifts, they

would appear to be exceptions to the above relationship.

However, there were several possible reasons for the small

Gpp(NH)p-induced shift in potency for these compounds. These

agonists could promote an extremely tight receptor-Ns complex

such that even in the presence of a guanine nucleotide little

complex destabilization (and hence a reduction in agonist

potency) is observed. If this is correct, then the loss of

Ns should reduce the potency beyond that observed in the

presence of Gpp(NH)p. However, this possibility was unlikely

since the IC50 values for Carbo-Am were very similar using

control reticulocyte membranes in the presence of Gpp(NH)p,

using reticulocyte membranes treated at 500C (to reduce

functional Ns) [Baker et al., 1985], and using erythrocyte

membranes (which lack Ns) [Limbird et al., 1980a]. An

alternative explanation for the small Gpp(NH)p-induced shift

could be an extremely tight (quasi-irreversible) or irre-

versible covalentt) binding of the carbostyril derivatives to

the receptor. Previous studies have shown that the major

effect of guanine nucleotides is to markedly increase the

dissociation rate of the agonist from the receptor (Williams

& Lefkowitz, 1977; Heidenreich et al., 1980). Thus an

extremely tight binding or covalent attachment of the agonist

to the receptor might greatly reduce or prevent the Gpp(NH)p-

induced agonist dissociation resulting in a small potency

shift. As discussed below, this was likely for the

carbostyril derivatives.

Several lines of evidence suggested that the carbostyril

derivatives bound in an extremely tight and/or irreversible

manner to the beta-adrenoreceptor. Scatchard analysis of

[125I]CYP binding after incubation with Carbo-Br or Carbo-Am

and membrane washing indicated a quasi-irreversible inter-

action as the receptor capacity decreased, whereas the KD for

[125I]CYP binding to the remaining receptors did not change.

In keeping with the small Gpp(NH)p-induced shifts to lower

potency for both compounds, the inclusion of Gpp(NH)p during

the membrane preincubation reduced the receptor loss to a

relatively small degree. Furthermore, the receptor loss

induced by the carbostyril derivatives was largely attenuated

by concurrent incubation with nadolol. These results

suggested that the initial interaction of both carbostyril

derivatives with the receptor was competitive followed by a

longer term quasi-irreversible binding. The Carbo-Am

compound contained no highly reactive moiety, suggesting that

its tight binding to the receptor was noncovalent in nature.

This was supported by the finding that a substantial reversal

of Carbo-Am binding occurred (>50%) by incubation of

membranes at 450C in the presence of nadolol. This effect

may have been due to a high temperature-induced dissociation

of the ligand from an intact receptor structure or a

dissociation induced by a reversible denaturation of the

receptor at 450C. A quasi-irreversible binding of two

beta-adrenoreceptor antagonists has been previously reported

(Terasaki et al., 1979; Lucas et al., 1979). In contrast to

the Carbo-Am, the Carbo-Br compound contained a reactive

bromoacetyl moiety. Thus its binding to the receptor may

have involved a loss of the bromo group resulting in a

reactive electrophilic ligand that could undergo an

irreversible alkylation of a nucleophile in the receptor.

This was supported by the observation that only a small

reversal of Carbo-Br binding (<10%) occurred by membrane

heating at 450C. Furthermore, after reacting Carbo-Br with

cysteine to inactivate the bromoacetyl moiety, a relatively

large reversal (58%) of its (carbo-cysteine) binding to the

receptor was found after membrane heating at 450C. Although

these data were consistent with a covalent attachment of

Carbo-Br to the receptor, another alternative was that Carbo-

Br bound reversibly with even higher affinity than Carbo-Am

such that little dissociation occurred, even at 450C.

Definitive proof of a covalent interaction would require more

experiments, perhaps using a radiolabelled compound in

conjunction with purified receptors as previously described

for an alkylating antagonist (Dickinson et al., 1985).

Interestingly, the maximal receptor loss induced by a wide

range of Carbo-Am concentrations was about 75%, suggesting

that a small fraction of receptors was resistant to the tight

binding of this compound. In contrast, the maximal receptor

loss induced by Carbo-Br approached 100%.

The data from the time course of reticulocyte adenylate

cyclase activation suggested that the two carbostyril

compounds acted as quasi-irreversible agonists. This was

indicated by the observation that addition of a 20-fold

excess of propranolol after seven min of incubation with the

two derivatives alone did not affect the rate of cAMP

accumulation. During the first seven min of incubation alone

with Carbo-Am or Carbo-Br, there was a 59 and 71% reduction

in specific [125I]CYP binding sites, respectively. In

contrast, when propranolol was added after seven min of

incubation with Iso alone, there was a complete blockade of

further cAMP production, consistent with Iso being a fully

reversible agonist. In addition, when propranolol was added

concurrently with both of the carbostyril compounds and Iso,

stimulation of the enzyme was virtually prevented. Thus the

cyclase activation data in conjunction with the previously

discussed binding data indicated that the initial interaction

of the carbostyril derivatives with the beta-adrenoreceptor

was competitive followed by a longer term quasi-irreversible

interaction. The activation of receptors by agonists has

been explained by two basic theories. The occupation theory

involved receptor activation as long as the receptor was

occupied whereas the rate theory predicted that activation

was proportional to the rate of combination between the

receptor and agonist (see Bowman & Rand, 1980; Yamamura et

al., 1985 for reviews). The data showing an apparent irre-

versible activation of adenylate cyclase by the carbostyril

congeners supported the occupancy theory for the beta-

adrenergic system.

In comparison to the apparent irreversible agonist

effects of the carbostyril derivatives, we recently reported

that bromoacetylaminomenthylnorepinephrine (BAAN) could,

under defined conditions, bind to the beta-adrenoreceptor in

an irreversible manner (Baker et al., 1985). Although BAAN

initially stimulated adenylate cyclase activity, after

irreversible binding it acted as an antagonist. The reasons

for the lack of an irreversible agonist effect by BAAN

whereas the carbostyril derivatives produced an apparent

irreversible agonist action were not obvious. These

differences might be related to the structures of the

compounds whereby different ligand-induced conformational

changes in the receptor were produced. Alternatively, the

differences might be related to the observation that BAAN was

a weak partial agonist whereas the carbostyril derivatives

were potent full agonists. Further studies would be neces-

sary to delineate the apparent irreversible effects of these

compounds. Although, to our knowledge, this was the first

report describing an apparent irreversible agonist for the

beta-adrenergic system, ligands showing irreversible or

sustained agonist effects have been reported for the insulin

(Brandenburg et al., 1980), adrenocorticotropin (Ramachandran

et al., 1981), adenosine (Lohse et al., 1986), and opiate

(Schoenecker et al., 1987) receptors.

45

In summary, the experimental results indicated that the

two carbostyril derivatives were stable, potent full beta-

adrenergic agonists that produced sustained activation

effects in vitro. The tight binding of the Carbo-Am to the

beta-adrenoreceptor appeared to be noncovalent whereas the

quasi-irreversible binding of the Carbo-Br might have

involved receptor alkylation.

CHAPTER 3
INTACT CELL STUDIES

Introduction

All of the experiments described thus far were performed

on rat reticulocyte membranes. This preparation offered a

simplified, cell-free system within which various components

could be easily manipulated. Some interesting results per-

taining to the interactions of the carbostyril congeners with

beta2-adrenoreceptors were obtained through examination of

this simplified system. Nonetheless, beta-adrenoreceptor

agonists and antagonists have been shown to interact in vivo

with living cells, not membrane fragments (Abramson &

Molinoff, 1984). Beta-adrenoreceptors on intact cells

possessed properties not found in simple membrane

preparations. One example of this was the agonist-induced

process of desensitization. The desensitization sequence

began with a rapid uncoupling of cell surface beta-adreno-

receptors from stimulation of adenylate cyclase (Su et al.,

1980; Harden et al., 1979). This was followed by an apparent

internalization or sequestration of the beta-adrenoreceptor

from the cell surface (Toews et al., 1984; Toews & Perkins,

1984; Harden et al., 1980), and eventual down-regulation

(loss of binding function) of total receptor number (Su et

al., 1980). The ability of internalized receptors to recycle

back to the cell surface depended on the length of exposure

to the agonist. In most cases (i.e., acute agonist

pretreatment), when the agonist was removed, receptors would

reappear on the cell surface without further protein

synthesis (Hertel & Staehelin, 1983; Stadel et al., 1983;

Doss et al., 1981). In contrast, after chronic agonist

treatment, receptors were internalized and degraded.

Therefore, because of the differences between an intact cell

system and isolated membranes, we have characterized the

agonist-induced changes of Carbo-Br in the DDT1-MF-2 smooth

muscle cell line. The ability of Carbo-Br to bind to alphal-

adrenoreceptors present in DDT membranes was examined also.

Evidence was provided to indicate that while Carbo-Br bound

either extremely tightly or irreversibly, it still caused a

desensitization effect in the same manner as Iso.

Experimental Procedures

Source of Materials

The radioligands (-)-[3H]CGP-12177 ([3H]CGP; 38.8-53.1

Ci/mmol) and [3H]Prazosin (82 Ci/mmol) were obtained from New

England Nuclear, Boston, MA, USA. The antagonist CGP-20712A

was a generous gift from Dr. L. Maitre at Ciba-Geigy, Ltd.,

Basle, Switzerland. Three-isobutyl-one-methylxanthine

(IBMX), and snake venom (crotalus atrox, western diamondback

rattlesnake) were obtained from Sigma Chemical Co., St.

Louis, MO, USA. Dowex 1-X8 anion exchange resin (200-400

mesh, chloride form) was purchased from Bio-Rad Laboratories,

Richmond, VA, USA and 2-mercaptoethanol was a gift from Dr.

S.R. Childers, University of Florida. Cultured cell lines

DDT1-MF-2 and C6 were obtained from American Type Culture

Collection, Rockville, MD, USA. The C62B cell line was a

gift from Dr. Mark Rasenick, University of Illinois College

of Medicine.

Methods

Buffer. Studies with intact cells were carried out

using Hank's Balanced Salt Solution (HBSS) unless otherwise

noted. The solution contains 5 mM KC1, 0.4 mM KH2P04, 137 mM

NaC1, 4 mM NaHCO3, 0.6 mM Na2HPO4, 6 mM D-glucose, 0.5 mM

MgCl2, 0.4 mM MgSO4, and 1 mM CaC12, pH 7.4.

Cell culture. The DDTI-MF-2 cell line (a smooth muscle

cell line derived from a leiomyosarcoma of the ductus def-

erens of Syrian hamster) and both lines of C6 cells (from rat

glial tumors) were grown in Dulbecco's modified Eagle's

medium supplemented with 5% fetal calf serum, 2.75 gg/ml

amphotericin B, 100 U/ml penicillin G, and 0.1 mg/ml strep-

tomycin sulfate in a humidified atmosphere of 95% air/5% C02.

Cells were routinely subcultured every seven days with

trypsin (0.01%) from an initial inoculum of 4-6 x 105 c/ml.

Cell harvesting and treatment. Cells were harvested at

approximately 50% confluence. Cell monolayers were washed

twice with PBS to remove culture medium. Cells used in

intact cell studies were incubated for 5 min at 360C in 3-10

ml of PBS containing 1 mM EGTA. The cells were lifted with

repeated pipetting, centrifuged at 1000xg for 5 min, and

resuspended in HBSS. In pretreatment studies, cells were

incubated for various times in the presence or absence of 10

gM Iso or 1 pM Carbo-Br (final concentrations) at 360C.

Cells were then washed five times with cold HBSS by centrif-

ugation at 1000xg followed by resuspension. Whole cell

homogenates were obtained by resuspending the final pellet in

50 mM Tris-HCl pH 7.4 containing 5 mM MgC12 and homogenizing

for 15 sec at setting 2.5. Binding assays were performed as

described above with [125I]CYP using 0.5 mg/ml protein.

Antagonist Binding Assays. Beta-adrenoreceptor content

of DDT and C6 membranes was determined by incubating membrane

protein (25 gg) in a total volume of 0.25 ml with 50 mM Tris-
HCl at pH 7.4, 5 mM MgCl2, 3-100 pM [125I]CYP and with and

without 1 JM ()alprenolol for 60 min at 360C. At the end of

the incubation, each suspension was diluted with 3 ml of 50

mM Tris-HCl pH 7.4 (360C) and poured onto a Whatman GF/B

glass fiber filter under reduced pressure. The filter was

washed with a further 6 ml of buffer, placed in a vial and

the radioactivity determined. Specific [125I]CYP binding to

the beta-adrenoreceptor was calculated as the difference

between the total binding in the absence of ()alprenolol and

the nonspecific binding determined in the presence of 1 gM

()alprenolol. Nonspecific binding was the same if

()alprenolol was replaced with 100 gM Iso. Specific binding

was 90-95% of the total bound.

In some experiments, the ability of the carbostyril

congeners, Iso, or the betal-selective antagonist CGP-20712A

to inhibit specific [125I]CYP binding was determined. Assays

were the same as above except the [125I]CYP concentration was

30 pM and 0.1% sodium ascorbate was included when Iso was

used. The competitive binding assays were also performed in

the presence and absence of 100 gM Gpp(NH)p, a non-hydro-

lyzable analog of GTP. All binding assays were performed in

triplicate, the results varying by less than 5%.

The recently introduced beta-antagonist CGP-20712A has

been shown to have an extremely high selectivity (about

10,000-fold) for the betal-adrenoreceptor subtype (Dooley &

Bittinger, 1984; Lemoine et al., 1985). In competition

assays with a nonselective labelled ligand, low concentra-

tions of CGP-20712A bound to the betal-subtype. As the

concentration of CGP-20712A was increased, a plateau region

appeared where no further displacement of labelled ligand

occurred until a high enough concentration of CGP-20712A was

present to displace binding of the labelled ligand to the

beta2-subtype. The ratio of betai to beta2 receptors could be

estimated from the plateau region of the inhibition curve or

calculated using various curve-fitting programs for the

analysis of multiple site binding data.

Beta-adrenoreceptor content on the surface of intact DDT

cells were measured by incubating cells (0.2 mg/tube) in a

total volume of 1.0 ml containing HBSS, 0.2 to 6.3 nM

[3H]CGP, and in the presence and absence of 1 JLM

()alprenolol for 2 hr at 40C. At the end of the incubation,

4 ml of 50 mM Tris-HCl pH 7.4 containing 5 mM MgC12 at 40C

was added to each tube, and the suspension was poured onto a

GF/C glass fiber filter under reduced pressure. Filters were

washed quickly with an additional 8 ml of the same buffer,

placed in scintillation vials with 7 ml of Liquiscint and the

radioactivity determined. Specific ligand binding was

calculated as previously described.

In some experiments, intact cells, in suspension (2

mg/ml) or plated on 143 cm2 plates, were pretreated for

various times with 10 gM Iso or 1 1M Carbo-Br at 360C. The

cells (or plates) were promptly removed to ice, washed five

times with HBSS at 40C, and resuspended in HBSS (cells) or

reinoculated in fresh media (plates). The suspended cells

were then assayed with a single concentration of [3H]CGP

(0.75 nM) as previously described.

Alphal-adrenoreceptors were measured by incubating

membrane protein (0.4 mg/tube) in a total volume of 2.0 ml of

50 mM Tris-HCl pH 7.4, containing 5 mM MgCl2, 0.25 nM

[3H]Prazosin, and in the presence or absence of 10 (M phent-

olamine for 30 min at 300C. At the end of the incubation, 4

ml of 50 mM Tris-HCl pH 7.4, containing 5 mM MgCl2 at 40C was

added to each tube, and the suspensions poured onto GF/C

glass fiber filters under reduced pressure. Filters were

washed quickly with an additional 8 ml of the same buffer,

placed in scintillation vials with 7 ml Liquiscint and the

radioactivity determined. Specific ligand binding was

determined as described earlier.

Measurement of cAMP production in intact cells. Intact

cell cAMP accumulation was measured by incubating detached

cells with various concentrations of Carbo-Br or Iso for 5

min at 360C in HBSS that contained 0.5 mM of the phospho-

diesterase inhibitor IBMX. At the end of the incubation,

cells were quickly (20 sec) sedimented by centrifugation at

1000xg to remove the drug as well as any extracellular cAMP.

The supernatant was aspirated, the pellet resuspended in 25

mM Tris-HCl buffer (pH 7.0, 40C), and samples placed in a

boiling water bath for 5 min. After boiling, samples were

centrifuged at 1000xg and the supernatant saved for cAMP

assay as described in Chapter two. For the time course of

cAMP accumulation or phosphodiesterase activity assay, cells

were incubated with either 1 iM Carbo-Br or 10 pM Iso for

various times in the absence of IBMX. The phosphodiesterase

inhibitor was added to some cell suspensions after the

incubation and before the first spin to prevent further cAMP

degradation.

DDT Membrane Adenylate Cyclase Assay. Activity was

determined as described earlier in Chapter two "Methods"

except that the membrane protein assayed was 65 ig/tube.

"Basal" conditions included membrane protein and the regen-

erating system which included ATP, creatine phosphokinase,

phosphocreatine, EGTA, BSA, and theophylline. "Carbo-Br"

conditions included basal additions plus GTP and Carbo-Br.

"Propranolol" conditions included Carbo-Br additions plus

propranolol. The cAMP content of the supernatant was

determined as described in Chapter two, "Methods".

Cyclic AMP Phosphodiesterase Assay. The activity of

cAMP phosphodiesterase was examined by utilizing the assay

procedure of Thompson & Appleman (1971) with modifications by

Meeker & Harden (1982). With all additions made on ice,

whole DDT cell homogenates (300 gg/200 .1) were added to

tubes containing 20 mM MgCl2, 4 mM 2-mercaptoethanol, 60 ig

bovine serum albumin, 0.5-400 gM cAMP, and 100,000 cpm/assay

of [3H]cAMP. All dilutions from stock were made with 40 mM

Tris-HCl (pH 7.4 at 300C). The total assay volume was 0.4

ml.

After incubation at 300C for 20 min, the reaction was

terminated by transferring the tubes to a boiling water bath

for 2 min. The tubes were then placed in a 40C bath where

100 p. of snake venom toxin (1 mg/ml) was added. The tubes

were incubated for another 10 min at 300C, and transferred

back to the 40C bath were the incubation was stopped by the

addition of 1.0 ml of a 1:2 slurry of Dowex 1-X8. The tubes

were mixed by vortexing and the Dowex sedimented by centrifu-

gation for 5 min at 1000xg. An aliquot of 0.5 ml was taken

from each tube, added to 9.5 ml Liquiscint and counted for 5

min. Blanks were determined in the presence of boiled

protein.

Data analysis. The competition data using CGP-20712A

were analyzed both as a one- and two-site model with a "By

Hand" curve-fitting program for the analysis of multiple site

radioligand binding data (Richardson & Humrich, 1984). This

analysis provided estimates of the competitor's affinity for

and the concentration of the two sites. The best fit of the

experimental data was obtained as defined by a minimum

deviation. The analysis assumed the ligand binding to both

sites followed mass-action kinetics. Arguments supporting

this assumption have been presented by Minneman et al.

(1979). Statistical analysis of all other data was performed

using the Student's t-test and was presented as mean S.E.

Results

Inhibition of specific f125I1CYP binding in DDT membranes

by Carbo-Br and Iso. Figure 3-1 showed the ability of Iso

and Carbo-Br to inhibit specific binding of [125I]CYP to DDT

cell membranes in the presence of 0.1 mM Gpp(NH)p. The IC50

values for Carbo-Br and Iso were 9 2 (n=3) and 400 23 nM

(n=3), respectively.

Effects of Carbo-Br on DDT cell cAMP production and

phosphodiesterase activity. Figure 3-2 showed the ability of

Iso and Carbo-Br to stimulate cAMP accumulation in intact DDT

cells. Carbo-Br was 9-fold more potent then Iso at stimu-

lating cAMP accumulation with an EC50 value of 9 1.6 nM

(n=3) compared to 80 12 nM (n=3) for Iso. A time course of

cAMP accumulation in DDT cells induced by 1 LM Carbo-Br or 10

AM Iso was shown in Figure 3-3. The time of cAMP

accumulation by both agonists was the same over the 60 min

time period. After 3 min of incubation there was an

o 100 a Iso
5 a Carbo-Br
80

60

o 40

u 20 -

-w 0 L -
-10 -9 -8 -7 -6 -5
Ligand (Log M)

Figure 3-1. Inhibition of specific [125I]CYP binding in DDT
cell membranes by Iso and Carbo-Br. Membranes (25 gg/tube)
were incubated with buffer at pH 7.4, 30 pM [125I]CYP, 100 IM
Gpp(NH)p, and the indicated concentrations of Iso and Carbo-
Br for 45 min at 360C. In the Iso competition assays, 0.1%
ascorbate was also present. At the end of the incubation,
the specific binding was determined as described under
"Methods". Each point on the graph was the mean of 3
determinations assayed in triplicate. Specific [125I]CYP
binding ranged from 63 to 69 fmol/mg protein.

.$120-
C L

90-

S60-

30-

o i
10 9 8 7 6 5
Agonist (-Log M)

Figure 3-2. Stimulation of cAMP production in intact DDT
cells by Iso and Carbo-Br. Cyclic AMP production was
determined by incubating intact cells (0.36 mg) in HBSS
buffer containing the indicated concentrations of Iso (closed
circles) or Carbo-Br (open circles) for 5 min at 360C. The
determination of cAMP content was performed as described
under "Methods". Each data point was the mean + S.E., n=3.
Basal activity was 10 pmol/min/mg protein and was subtracted
from the stimulated values.

-~. 60

2 50-
0)
E 40

E
- 30-

E 20-
O T
S1 0 l1 *

0 10 20 30 40 50 60
Time (min)

Figure 3-3. Time course of cAMP accumulation in intact DDT
cells by Iso and Carbo-Br. Cyclic AMP accumulation was
determined by incubating intact cells (0.36 mg) in HBSS
buffer containing 10 gM Iso (closed squares) or 1 JiM Carbo-Br
(open squares) at 360C for the indicated times. Determin-
ation of cAMP was performed as described under "Methods".
Each data point was the mean of 3-4 determinations S.E.
Basal cAMP formation (10 pmol/min/mg protein) has been
subtracted from the stimulated values.

80-

0
- 60
0)
E

E 40

S 20-

a.

o 0-
0 10 20 30 40 50 60
Time (min)

Figure 3-4. Time course of cAMP accumulation in intact DDT
cells by Iso and Carbo-Br after addition of propranolol.
Cyclic AMP accumulation was determined by incubating intact
cells (0.36 mg) in HBSS buffer containing 10 gM Iso (closed
squares) or 1 JM Carbo-Br (open squares) plus 20 pM propran-
olol added to all tubes after 3 min of incubation at 360C.
Determination of cAMP was performed as described under
"Methods". Each data point was the mean of 3-4 determin-
ations S.E. Basal cAMP formation has been subtracted from
stimulated values.

Intact cells (2.4 mg) were incubated for various times
in HBSS buffer pH 7.4 containing 1 JLM Carbo-Br or 10 LM Iso
at 360C. At the end of the incubations, cells were
sedimented by centrifugation at 1000xg and resuspension in
0.9 ml of40 mM Tris-HCl. Cells were then homogenized, and
the whole cell homogenate (0.3 mg/tube) assayed for
phosphodiesterase activity as described in "Methods".

Intact cells (1.5 mg) were incubated for various times in
HBSS buffer at pH 7.4 in the presence or absence of 1 JM Carbo-Br.
At the end of the incubations, the cells were washed four times by
centrifugation at 1,000xg and resuspension. After the final wash,
cells were resuspended in 50 mM Tris-HC1 pH 7.4, homogenized, and
the membranes sedimented by centrifugation at 35,000xg for 10 min.
DDT cell membranes (65 gg/tube) were assayed for adenylate cyclase
activity as described in "Methods", and the results were listed
under the appropriate assay condition heading.

a Significantly different from the respective control, p 0.005;
b Significantly different from the respective control, p 0.05;
c Significantly different from the respective control, p < 0.01,
as determined by unpaired student's t-test.
All values were means S.E., n=3-4.

basal activity declined in membranes from Carbo-Br treated

cells over an hr, from 2.5-fold at 3 min to 1.4-fold

stimulation at 60 min.

It was important to notice also that propranolol blocked

Carbo-Br stimulated cAMP production in control membranes when

the two were added together, but had no effect on cAMP

production in membranes from cells that were pretreated with

Carbo-Br (Table 3-2). This was consistent with our findings

in reticulocytes and whole cells that Carbo-Br had bound

tightly and/or irreversibly by at least 3 min, yet still

stimulated cAMP production.

Effects of Carbo-Br and Iso pretreatment on DDT cell

beta-adrenoreceptors. Receptor content was assayed in two

ways: whole cell homogenates were assayed using [125I]CYP,

and intact cells were assayed using the hydrophilic beta-

antagonist [3H]CGP which measured only those receptors found

on the cell surface (Hertel et al., 1983; Wilkinson &

Wilkinson, 1985). Figure 3-5 was a representative Scatchard

plot of [125I]CYP binding in whole cell homogenates after a 3

min pretreatment of intact cells with 1 LM Carbo-Br, 10 LM

Iso or buffer alone followed by cell washing. The slopes of

the lines were parallel, indicating that all of the unbound

drugs have washed out (KD values: control, 22 4; Iso-

treated, 16 3; Carbo-Br-treated, 29 8 pM, n=4-7). There

was a 50% loss of binding sites after only a 3 min

pretreatment with Carbo-Br (Bmax values: control, 58 3,

Iso-treated, 57 6; Carbo-Br-treated, 34 5 fmol/mg

5.0

o
o 4.0 -
o

3.0

2.0
C !
0 A
00 1.0

0.0
0 10 20 30 40 50 60 70

[1251]CYP Bound (fmol/mg protein)

Figure 3-5. Scatchard plot of specific [125I]CYP binding to
whole DDT cell homogenates after intact cell treatment with
Iso and Carbo-Br. Intact cells (3 mg) were incubated in HBSS
buffer containing 10 pM Iso (triangles), 1 pM Carbo-Br
(closed squares) or buffer alone (open squares) at 360C for 3
min. Samples were removed, intact cells washed five times
with ice-cold HBSS buffer, resuspended in 50 mM Tris
containing 5 mM MgCl2, and homogenized. The homogenates were
assayed with 3 to 100 pM [125I]CYP as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (pmol/mg protein) to free ligand
(pmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.

= 100

80
o 80

60

o 40 -

20

0
0 10 20 30 40 50 60
Time (min)

Figure 3-6. Time course of specific [125I]CYP binding loss
in whole DDT cell homogenates after intact cell treatment by
Iso and Carbo-Br. Intact cells (3 mg) were incubated in HBSS
buffer containing 10 LM Iso (closed squares), 1 JM Carbo-Br
(open squares), or buffer alone (circles) at 360C. At the
times indicated, samples were removed, intact cells washed
five times with ice-cold HBSS buffer, resuspended in 50 mM
Tris-HCl containing 5 mM MgC12 and homogenized. The homog-
enates were assayed with 75 pM [125I]CYP as described under
"Methods". Data points were the mean of four determinations.
The control [125I]CYP values were 48 4 fmol/mg protein.

Figure 3-7. Time course of specific [3H]CGP binding loss by
Iso and Carbo-Br in intact DDT cells. Intact cells (6 mg)
were incubated in HBSS buffer containing 10 pM Iso or 1 gM
Carbo-Br at 360C. At the times indicated, samples were
removed to ice, intact cells washed five times with ice-cold
HBSS buffer, resuspended in 40C HBSS and assayed with 0.75 nM
[3H]CGP as described under "Methods". Data points were the
mean of three determinations (significantly different from
control, *p 0.005; **p 5 0.05). The control [3H]CGP binding
values ranged from 60.2 to 69.6 fmol/mg protein.

90-
Control
U Isoproterenol

c 60
-I

E
6 30

0

1 24
Time (hours)

Figure 3-8. Loss of specific [3H]CGP binding in intact DDT
cells immediately or 24 hr after a 60 min treatment with Iso
or Carbo-Br. DDT cells plated on 143 cm2 plates were
incubated in HBSS buffer containing 10 lM Iso or 1 LM Carbo-
Br for 60 min at 360C. At that time, the plates were removed
to 4C and washed five times with ice-cold HBSS buffer. One-
half the cells from each treatment group were given DMEM plus
5% FCS and removed to the cell incubator. The remaining
cells were lifted with ice-cold HBSS containing 1 mM EGTA,
resuspended in 40C HBSS and assayed with 0.75 nM [3H]CGP as
described under "Methods". Twenty-four hr later the rest of
the cells were lifted and assayed as the former. Data points
were the mean of three determinations (significantly dif-
ferent from control; *p < 0.0005, **p 5 0.025). The control
[3H]CGP binding values were 74.3 1.63 fmol/mg protein for 1
hr and 76.7 0.76 for 24 hr (mean + S.E.).

Table 3-3. Loss of binding sites in two C6 clones after
Carbo-Br pretreatment

Bmax (fmol/mg protein)

Cell line Control Carbo-Br

C6 33.3 0.1 25.2 1.5a

C62B 16.3 1.4 13.6 1.6

Cell membranes (4.0 mg) were preincubated in the
presence or absence of 1 1M Carbo-Br for 15 min at 360C in 50
mM Tris-HC1 buffer pH 7.4 containing 5 mM MgCl2 and 100 M
Gpp(NH)p. At the end of that time, membranes were washed 5
times by centrifugation at 35,000xg and resuspension.
Membranes (25 gg/tube) were assayed with 3-100 pM [125I]CYP
as described in "Methods".

a Significantly different from control, p 5 0.025, as
determined by unpaired student's t-test.

All values given as means S.E., n=3.

69

S100

80

60
60

o 40

Control
S20 Carbo-Br

P "
0 i I I I
-11 -10 -9 -8 -7 -6 -5 -4

Log [CGP-20712AR

Figure 3-9. Inhibition of specific [125I]CYP binding in C6
cell membranes by CGP-20712A after Carbo-Br treatment.
Membranes (4.0 mg)were incubated with buffer at pH 7.4 in the
presence or absence of 1 JLM Carbo-Br for 15 min at 360C. The
membranes were washed five times with ice-cold buffer and
assayed with 30 pM [125I]CYP, 100 gM Gpp(NH)p, and the
indicated concentrations of CGP-20712A for 45 min at 360C.
The curves were representative of three experiments.
Specific [125I]CYP binding totals were 18.6 and 13.8 fmol/mg
protein for control and Carbo-Br treated membranes,
respectively.

Figure 4-2. Inhibition of specific [125I]CYP and [3H]DHA
binding in rat tissue membranes by CGP-20712A following in
vivo Carbo-Br treatment. A) Heart (squares), spleen
(circles); B) sumaxillary gland (squares), lung (circles).
Male rats were injected i.p. with 5 mg/kg Carbo-Br (closed
symbols) or vehicle (open symbols). After 3 hr, submaxillary
glands, hearts, lungs, and spleens were removed. Membranes
were prepared and assayed with 30 pM [125I]CYP or 5 nM
[3H]DHA, and the indicated concentrations of CGP-20712A for
45 min at 360C. At the end of the incubations, specific
binding was determined as described under "Methods". Each
point on the graph was the mean of four determinations
assayed in triplicate. The control [125I]CYP binding values
were 48 2, 14.5 0.5, and 50 2.6 fmol/mg protein for the
submaxillary gland, heart, and spleen, respectively. The
control [3H]DHA binding value for the lung was 343 28
fmol/mg protein. Specific [125I]CYP binding values for
Carbo-Br treated tissues were submaxillary gland, 37.5 1.8;
heart, 8.8 0.25; and spleen, 6.2 0.3 fmol/mg protein.
Specific [3H]DHA binding for the Carbo-Br treated lung tissue
was 76.6 6.3 fmol/mg protein. Each value was the mean of
six to eight determinations.

C

E 20-

H
c- O
0

c 100-
a B
o

c
S80
CD
' 60

40-

20-

I I
10 9 8 7 6
CGP-20712A (-Log M)

Table 4-1. Concentration of Iso and Carbo-Br that inhibit
specific ligand binding to the beta-adrenoreceptors from
several rat tissues by 50% (IC50)

IC50 nM a

Tissue Iso Carbo-Br

Heart 443 55 95 7b

Submaxillary gland 449 41 96 llb

Spleen 613 23 23 6b

Lung 543 22 24 5b

Membranes from each tissue were incubated with 50 mM
Tris-HCl buffer at pH 7.4 containing 5 mM MgCl2, various
concentrations of Iso or Carbo-Br, and 100 IM Gpp(NH)p for 45
min at 360C. The incubations also included 30 pM [125I]CYP
for heart, submaxillary gland, and spleen assays, and 5 nM
[3H]DHA for lung assays. The total specific binding values
were similar to those given as control values in Figure 4-2.

a Each value was the mean S.E., n= 4-8.
b Significantly different from Iso values, p < 0.0005 as
determined with unpaired student's t-test.

control, there was a 71% decrease in specific [3H]DHA binding

with no effect of Gpp(NH)p on receptor loss. In addition,

there was no change in the KD value for [3H]DHA binding to the

remaining receptors after Carbo-Br pretreatment as compared

to the control (control, 0.85 nM; Carbo-Br pretreated, 0.74

nM; Carbo-Br plus Gpp(NH)p pretreated, 0.87 nM). Figure 4-4

showed the same experimental protocol using cardiac ventric-

ular membranes. After pretreatment with Carbo-Br or Carbo-Br

plus Gpp(NH)p there was a 30 and 15% loss of specific

[125I]CYP binding sites, respectively. However, there was an

increase in the KD value for [125I]CYP binding (control, 20

pM; Carbo-Br pretreated, 38 pM; Carbo-Br plus Gpp(NH)p

pretreated, 38 pM).

Physiological effects of Carbo-Br. Figures 4-5 and 4-6

showed the ability of Iso and Carbo-Br to stimulate lung and

heart ODC activity, respectively. Three hours after a 0.1

mg/kg injection of Iso there was an 8.0- and 8.6-fold

increase in lung and heart ODC activity, respectively.

Increasing the dose of Iso to 5 mg/kg increased the fold

stimulation to 21- for both tissues. At a dose of 0.1 mg/kg

of Carbo-Br in both tissues, there was an 8-fold stimulation

of enzyme activity that was increased to 12-fold 3 hr after a

5 mg/kg dose. When propranolol (20 mg/kg) was given 30 min

before the 5 mg/kg dose of either agonist, enzyme stimulation

was completely blocked in both tissues. Finally, Figures 4-5

0.8

S 0.6- 03

g 0.4- [

II !
1 0.2

0 100 200 300 400 500 600

[3HIDHR Bound (fmol/mg protein)

Figure 4-3. Scatchard plot of specific (3H]DHA binding to
rat lung membranes after treatment with Carbo-Br in vitro.
Membranes were incubated without (open squares), with 0.5 LM
Carbo-Br (closed squares), and with 0.5 gM Carbo-Br plus 100
JM Gpp(NH)p (triangles) for 30 min at 300C. At the end of
the incubation, the membranes were washed four times with
buffer and assayed with 0.31-10 nM [3H]DHA as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (pmol/mg protein) to free ligand
(pmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.

0.6

o 0.5

0.4 -

S0.3
u_

i 0.2

0.1

0 2 4 6 8 10 12 14 16
[1251]CYP Bound (fmol/mg protein)

Figure 4-4. Scatchard plot of specific [125I]CYP binding to
rat heart membranes after treatment with Carbo-Br in vitro.
Membranes were incubated without (open squares), with 0.5 gLM
Carbo-Br (closed squares), and with 0.5 pM Carbo-Br plus 100
pM Gpp(NH)p (triangles) for 30 min at 300C. At the end of
the incubation, membranes were washed four times with buffer
and assayed with 3 to 100 pM [125I]CYP as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (fmol/mg protein) to free ligand
(fmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.

40 -
40- basal
,c Lung isoproterenol

S30 Carbo-Br

E

20
-a

0 10 *
0

basal 0.1 mg 5 mg 5+P 5/24

Figure 4-5. Stimulation of ornithine decarboxylase activity
in rat lung 3 and 24 hr after in vivo Iso and Carbo-Br
treatment. Male rats were injected i.p. with 0.1 or 5 mg/kg
Iso or Carbo-Br, 20 mg/kg propranolol followed by 5 mg/kg Iso
or Carbo-Br, or with vehicle alone. After 3 or 24 hr, lungs
were removed and assayed for ODC activity as described in
"Methods". Each response was the mean of three determin-
ations assayed in triplicate (significantly different from
basal levels, *p 5 0.0005).

30 -
30 Heart M Basal
-E Isoproterenol
E L Carbo-Br
0o
E 20

om 10
o *

O-

Basal 0.1 mg 5 mg 5+P 5/24

Figure 4-6. Stimulation of ornithine decarboxylase activity
in rat heart 3 and 24 hr after in vivo Iso and Carbo-Br
treatment. Male rats were injected i.p. with 0.1 or 5 mg/kg
Iso or Carbo-Br, 20 mg/kg propranolol followed by 5 mg/kg Iso
or Carbo-Br, or with vehicle alone. After 3 or 24 hr, hearts
were removed and assayed for ODC activity as described in
"Methods". Each response was the mean of three determin-
ations assayed in triplicate (significantly different from
basal levels, *p < 0.0005).